Throughout this document various references are referred to by their author and year of publication. Full citations for these references can be found immediately preceding the sequence listing. The contents of each of these references is hereby incorporated by reference in order to more fully describe the state of the art to which the subject invention pertains.
The significant role of platelet activation in hemostasis is well-documented. Platelet activation is necessary for platelet aggregation and secretion, and is initiated by the binding of agohist to receptors at the platelet surface. Over the last ten years, several laboratories have developed monoclonal antibodies to platelet membrane glycoproteins. These platelet membrane glycoproteins can serve as agohist receptors on the platelet membrane. The antibodies to these glycoproteins have been of great value in studies designed to elucidate the structure and function of these glycoproteins.
Most of the success in raising monoclonal antibodies to platelet receptors was in studies on the integrin cohesion receptor IIb/IIIa (Coller et al., 1983; Kornecki et al., 1984) and the adhesion receptor Ib/IX (Coller et al., 1983; Handa et al., 1986). Functional antibodies that inhibit the action of these receptors provided a large body of new information and have led to direct conclusions about the functions of these glycoprotein receptors. Such inhibitory antibodies were also shown to have potential in vivo therapeutic use (Collet et al., 1986; Peters et al., 1986).
Some of these antibodies that serve as agonists, binding to the receptors at the platelet surface and thereby activating the platelets, have been identified. Several laboratories have developed or identified such "activator" antibodies that appear to react with platelet membrane protein components of 21-24 kD. The first report of a monoclonal antibody which served as an agonist and induced platelet aggregation was published by Boucheix et al. in 1983. This monoclonal antibody immunoprecipitated a platelet protein with apparent molecular weight (M.W.) of 24 kD under both reduced and non-reduced conditions. The addition of Fab fragments of this antibody to platelets resulted in the inhibition of platelet aggregation induced by various agonists. The platelet antigen recognized by this antibody was identical to the leukemia-associated antigen, p24, found in common acute lymphoblastic leukemia cells and neuroblastoma cells (Kersey et al., 1981; Jones et al., 1982; Komada et al., 1983).
Thiagarajan et al. (1983) reported that platelet aggregation could be induced by another monoclonal antibody. This antibody was found to be directed against a 21 kD protein present in both normal and Glanzmann's thrombasthenic platelets.
Gorman et al. (1985) have described several monoclonal antibodies which induce platelet aggregation. All of these antibodies immunoprecipitated a 24 kD platelet protein in both the reduced and non-reduced states. The Fab fragments of these antibodies were found to augment the aggregation of platelets by adenosine diphosphate (ADP).
Higashihara et al. (1985) also described a monoclonal antibody which induced platelet aggregation and secretion by interaction with a protein of 24 kD. Preincubation of platelets with this antibody inhibited ristocetin-induced agglutination. It is known that these antibodies are directed against the p24/CD9 protein on the platelet surface. The CD9 antigen has been cloned and sequenced (Boucheix et al., 1991; Lanza et al., 1991), and CD9 antibodies have been shown to induce platelet aggregation mediated by the Fc.gamma.RII receptor (CD32 molecule) (Worthington et al., 1990).
Duncan and Rosse (1986) showed that antibodies to platelet HLA class I antigen (anti-ABH IgG) could activate platelets and induce serotonin release. Similar results were obtained by Cosgrove et al. (1988), who reported that three different anti-HLA Class I monoclonal antibodies and an anti-.beta..sub.2 microglobulin antibody caused platelet aggregation and secretion. Duncan and Rosse (1986) also showed that high concentrations of anti-Pl.sup.A1 antibodies inhibited platelet secretion induced by these antibodies. Ryu et al. (1989) found that high concentrations of PL.sup.A1 blocked fibrinogen binding resulting in the blockage of agohist-induced platelet aggregation, whereas low concentrations of anti-PL.sup.A1 B antibodies induced release and aggregation.
Activator monoclonal antibodies directed against GPIIb and GPIIIa have also been reported. A stimulatory monoclonal antibody to the GPIIb/IIIa complex has been described by Modderman et al. (1988) which induces the release of alpha and dense granule contents resulting in platelet aggregation. Morel et al. (1989) have described a monoclonal antibody directed against GPIIb. The F(ab').sub.2 fragments of this antibody did not induce platelet aggregation although they blocked the stimulation of platelets by the intact antibody.
In addition to these antibodies, antibodies of other specificity have been described which activate platelets. Scott et al. (1989) described a monoclonal antibody which stimulates platelet secretion and aggregation and is directed against a platelet membrane glycoprotein of M.W. 67 kD. Recently, Yanabu et al. (1991) detected an autoantibody in a patient with immunothrombocytopenia (ITP), which activated normal platelets by interacting with a 36 kD platelet surface protein.
Kornecki et al. (1990) referred to a monoclonal antibody called M.Ab.F11 which induces vesicular secretion and aggregation in human platelets.
The health related significance of these antibodies which can activate human platelets is apparent. Characterization of the antigens which serve as receptors for these antibodies in the activation process is necessary as well as the elucidation of the biochemical pathways triggered by these interactions.